Known scanning laser microscopes include an illumination arm and a detection arm. The illumination arm generates a focused probe beam which is scanned across a sample or object. The reflected, scattered, or emitted light is collected and detected synchronously with the scanning to build up an image on a pixel-by-pixel basis. The detection arm of the microscope may include spectral filters (for fluorescence or photoluminance imaging), a spatial filter (for confocal imaging) and/or a polarization analyzer (for polarization imaging).
The detection arm of the scanning laser microscope includes an objective lens that collects the diffracted light from the illuminated spot. The objective lens collects light diffracted from low spatial frequency features, in other words from relatively large and slowly changing features of the object within the area of the incident focused spot. Light is also diffracted from high spatial frequency features, which are defined as rapidly changing features of the object within the area of the focused spot. The light from the high spatial frequency features is scattered over high angles, which are outside of the diameter of the objective lens, and therefore not collected.
In a conventional scanning laser microscope the total beam incident on the detector for each pixel is detected. This amounts to spatial integration of the detected beam. The content of that detected beam is limited by the ability of the objective lens to collect light diffracted from the object. The classical diffraction limit, which determines the resolution of the microscope, is based on integrating the collected light.
In some known scanning laser microscopes, a Hartmann-Shack wavefront sensor (HSWS), has been added to measure the wavefront shape of the beam in order to correct for optical aberrations. The Hartmann-Shack wavefront sensor includes a micro lens array and a sensor array such as a CCD camera. Each lens of the micro lens array samples the incident field and focuses a spot on a portion of the sensor array. The intensity of each spot detected by the sensor array indicates the intensity of the sampled field, and the displacement of each spot indicates the gradient of the phase of the field in that sub-region (i.e. the local wavefront slope). The wavefront shape information from the Hartmann-Shack wavefront sensor is used to drive deformable mirrors in the illumination arm of the microscope in order to reduce any imaging aberrations that are present. Wavefront sensors and deformable mirrors can be used to advantage in situations where the sample is imaged through a distorting medium. An example is where the object to be imaged is beneath a thick glass plate which introduces spherical aberrations. Other applications have included astronomy and ophthalmology.